Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

Abstract

To investigate the regulation of tumor suppressor XAF1 gene expression in digestive system cancers, we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines (human hepatoma cell line HepG2, human colon cancer cell line LoVo, and human gastric cancer cell line AGS) and nontumor cell lines (human embryonic liver cell line L02 (L02 cells) and human embryonic kidney 293 cells [HEK293 cells]) as controls. 1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction (PCR) and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity. The plasmids were transfected into a variety of cell lines by lipofectamine 2000. The promoter transcription activity was determined by dual-luciferase report assay, and enhanced green fluorescent protein (EGFP)-positive cells were detected by fluorescence microscope. The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines. The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells. Expression of green fluorescent protein (GFP) under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells. The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells. The results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.

Keywords

gene / X-linked inhibitor of apoptosis protein associated factor-1 (XAF1) / promoter / transcription regulation

参考文献

[1] JacobsonM D, WeilM, RaffM C. Programmed cell death in animal development. Cell, 1997, 88(3): 347-354

[2] HorvitzH R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res, 1999, 59(7): 1701-1706

[3] ThompsonC B. Apoptosis in the pathogenesis and treatment of disease. Science, 1995, 267(5203): 1456-1462

[4] HanahanD, WeinbergR A. The hallmarks of cancer. Cell, 2000, 100(1): 57-70

[5] DeverauxQ L, ReedJ C. IAP family proteins-suppressors of apoptosis. Genes Dev, 1999, 13(3): 239-252

[6] FongW G, ListonP, Rajcan-Separovic E, St Jean M, Craig C, Korneluk R G. Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines. Genomics, 2000, 70(1): 113-122

[7] ListonP, FongW G, KellyN L, TojiS, MiyazakiT, ConteD, TamaiK, CraigC G, McBurneyM W, KornelukR G. Identification of XAF1 as an antagonist of XIAP anti-caspase activity. Nat Cell Biol, 2001, 3(2): 128-133

[8] PlenchetteS, CheungH H, FongW G, LaCasseE C, KornelukR G. The role of XAF1 in cancer. Curr Opin Investig Drugs, 2007, 8(6): 469-476

[9] ByunD S, ChoK, RyuB K, LeeM G, KangM J, KimH R, ChiS G. Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res, 2003, 63(21): 7068-7075

[10] NgK C, CamposE I, MartinkaM, LiG. XAF1 expression is significantly reduced in human melanoma. J Invest Dermatol, 2004, 123(6): 1127-1134

[11] GreenM, SchuetzT J, SullivanE K, KingstonR E. A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function. Mol Cell Biol, 1995, 15(6): 3354-3362

[12] WangJ, HeH, YuL, XiaH H, LinM C, GuQ, LiM, ZouB, AnX, JiangB, KungH F, WongB C. HSF1 down-regulates XAF1 through transcriptional regulation. J Biol Chem, 2006, 281(5): 2451-2459

[13] SmaleS T. Core promoters: active contributors to combinatorial gene expression. Genes Dev, 2001, 15(19): 2503-2508

[14] NaylorL H. Reporter gene technology: The future looks bright. Biochem Pharmacol, 1999, 58(5): 749-757

[15] JangJ Y, KimH J, ChiS G, LeeK Y, NamK D, KimN H, LeeS K, JooK R, DongS H, KimB H, ChangY W, LeeJ I, ChangR. Frequent epigenetic inactivation of XAF1 by promoter hypermethylation in human colon cancers. Korean J Gastroenterol, 2005, 45(4): 285-293

[16] CarperS W, DuffyJ J, GernerE W. Heat shock proteins in thermotolerance and other cellular processes. Cancer Res, 1987, 47(20): 5249-5255

[17] HightowerL E. Heat shock, stress proteins, chaperones and proteotoxieity. Cell, 1991, 66(2): 191-197

[18] TrautingerF, Kindås-MüggeI, KnoblerR M, HönigsmannH. Stress proteins in the cellular response to ultraviolet radiation. J Photochem Photobiol B, 1996, 35(3): 141-148

[19] SzatrowskiT P, NathanC F. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res, 1991, 51(3): 794-798

[20] HilemanE O, LiuJ, AlbitarM, KeatingM J, HuangP. Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity. Cancer Chemother Pharmacol, 2004, 53(3): 209-219

[21] WangD, KreutzerD A, EssigmannJ M. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat Res, 1998, 400(1,2): 99-115

[22] WeiH. Activation of oncogenes and/or inactivation of anti-oncogenes by reactive oxygen species. Med Hypotheses, 1992, 39(3): 267-270

[23] KroegerP E, MorimotoR I. Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity. Mol Cell Biol, 1994, 14(11): 7592-7603

[24] WangY, MorganW D. Cooperative interaction of human HSF1 heat shock transcription factor with promoter DNA. Nucleic Acids Res, 1994, 22(15): 3113-3118

[25] Birch-MachinI, GaoS, HuenD, McGirrR, WhiteR A, RussellS. Genomic analysis of heat-shock factor targets in Drosophila. Genome Biol, 2005, 6(7): R63

版权

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg